PyroMark Q24 Advanced

For advanced methylation and mutation quantification in long sequence runs using Pyrosequencing

Advanced technology, software, and chemistry for long sequence runs

Quantitative methylation analysis at consecutive CpG or CpN sites

Improved quantification of sequence variations at any sequence position

Easy and improved base calling functionality

Assay versatility on the same instrument and in the same run

PyroMark Q24 Advanced has improved Pyrosequencing technology to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced features advanced technology, software, and chemistry, and is highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites, complex mutations, or for de novo sequencing applications such as microbial typing.

PyroMark Q24 Advanced and PyroMark Q24 Advanced Software are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q24 Advanced for the applications described in the kit handbooks. If PyroMark Q24 Advanced and PyroMark Q24 Advanced Software are used with other than QIAGEN kits, it is the user's responsibility to validate the performance of such product combination for any particular application.

PyroMark Q24 Advanced increases both read length and reliability of methylation analysis at positions later in the sequence. This example demonstrates 135 nucleotide dispensations and the accurate analysis of 16 different CpG positions in a single PyroMark Q24 Advanced CpG reaction. To accurately analyze all of these sites with PyroMark Q24, one would need to run 3 separate assays.|A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin.|The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide.|ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.|Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.|Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace.|Quantification of CpG methylation directly following a T homopolymer or between T homopolymers is especially challenging. Methylation levels are determined by the ratio of converted C nucleotides to unconverted C nucleotides following bisulfite treatment, and since the C to T conversion often leads to long T homopolymer stretches in an amplicon, this scenario occurs often. PyroMark Q24 Advanced enables accurate quantification of CpG position after or between T homopolymers.This example shows the analysis of a CpG site within a stretch of 8 T nucleotides.|Read length is a critical factor for analysis of unknown sequences, often limiting the reliable analysis to 40–80 bases. The improved chemistry and algorithm of PyroMark Q24 Advanced significantly increases the possible read length and provides higher accuracy. PyroMark Q24 (upper panel) and PyroMark Q24 Advanced (lower panel) were used for de novo sequencing using the same assay. Blue bars indicate reliably detected bases; yellow indicates bases that might have been determined correctly but should be checked by the user; red indicates unreliable readings.|Since mutations and single nucleotide polymorphisms (SNPs) are rarely in directly neighboring positions, common Pyrosequencing chemistry might require separate assays for analysis of more than one mutation site. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of additional mutations within one run. This example shows the analysis of a 10:90 mixture of wild-type and mutated EGFR. Even after 60 dispensations, the analysis is exact.|The components of the PyroMark Q24 Advanced system are designed to make the Pyrosequencing workflow straightforward and efficient. Each step, from assay design to PCR amplification and preparation of sequencing templates, is supported by software, kits, reagents, and sample prep instrumentation optimized for Pyrosequencing.|Though small in size, PyroMark Q24 Advanced manages all steps necessary to rapidly analyze up to 24 samples. Simply load your samples and reagents, upload your run file, and walk away. The instrument dispenses reagents and nucleotides to each well with precision. Light signals emitted are detected by 24 CCD sensors — one sensor per well — thereby eliminating signal crossover.|PyroMark Q24 Advanced is designed as a stand-alone instrument, which makes it easy to place anywhere in the lab. Data are stored on the instrument hard drive and can be viewed on the instrument screen during a run. Additionally, all files are stored on the supplied USB stick, giving the user the flexibility to analyze data on any computer with PyroMark Q24 Advanced Software installed.|The PyroMark Q24 Vacuum Workstation enables conversion of PCR products into the single-stranded DNA for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process is carried out for 24 samples in parallel and takes only a few minutes.|

Longer Pyrosequencing runs and improved accuracy

PyroMark Q24 Advanced features improved chemistry and instrument operation algorithms that significantly increase the assay read length and accuracy of the base calling functionality, enabling easy analysis of long de novo sequencing runs. Assay read length was previously limited by background peaks and reduced light signals in the sequencing reaction. The updated chemistry and algorithms of PyroMark Q24 Advanced reduce these background peaks, thereby increasing read length and reliability. Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single PyroMark Q24 Advanced reaction (see figure Long de novo sequencing runs).

Improved methylation analysis at any position

PyroMark Q24 Advanced enables improved methylation quantification in long sequence runs at any sequence position. Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy, methylation quantification is highly reliable throughout the entire sequencing run (see figure Analysis of 16 CpG sites in a long sequence run).

Improved sequencing accuracy in homopolymers

Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Improved methylation quantification in homopolymers).

Analysis of mutations over long sequences

PyroMark Q24 Advanced also provides reliable quantification of multiple polymorphisms in a single assay. Since single nucleotide polymorphisms (SNPs) and other mutations are often not located close to one another, traditional Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one mutation or SNP in the same run (see figure Quantitative mutation analysis in long sequence runs).

Principle

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 Advanced is a fully integrated system that provides real-time sequence information with high accuracy and long read lengths, making it highly suitable for analysis of complex mutations, epigenetic research, resistance typing, and microbial identification. The system comprises the PyroMark Q24 instrument (running firmware 3.0 or higher), PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents/PyroMark Q24 Advanced CpG Reagents, PyroMark Q24 Vacuum Workstation, and PyroMark Control Oligo (see table). Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q24 Vacuum Workstation.

Enzymes, substrates, nucleotides, and buffers. Compatible only with assays set up and analyzed with PyroMark Q24 Advanced Software.

PyroMark Q24 Advanced CpG Reagents

Enzymes, substrates, nucleotides, and buffers for assays requiring longer sequence read lengths. Compatible only with assays set up and analyzed with PyroMark Q24 Advanced Software.

PyroMark Q24 Vacuum Workstation

Workstation for preparing up to 24 samples in parallel from PCR product to single-stranded template

PyroMark Q24 Control Oligo

Control for verification of proper installation and operation of the system

PyroMark Q24 Advanced system.

Steps of the Pyrosequencing reaction:

Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure "Principle of Pyrosequencing — step 1").
Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure "Principle of Pyrosequencing — step 2").

Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure "Principle of Pyrosequencing — step 3").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure "Principle of Pyrosequencing — step 5").

Streamlined workflow — from sample to result

The versatile PyroMark Q24 Advanced seamlessly integrates into epigenetic and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. The highly reliable instrument enables sequence-based detection and quantification of methylation at CpG or CpN sites, as well mutations. The streamlined workflow means that results can be achieved faster.

Procedure

Fast and easy sample preparation

From PCR product to single-stranded template ready for sequencing — up to 24 samples can be prepared in parallel using the PyroMark Q24 Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling, and the actual "hands-on time" is less than 5 minutes.

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. Whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, or polymorphisms in forensic samples of mitochondrial DNA, the PyroMark Q24 Advanced enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.

Easy-to-use PyroMark Q24 Advanced Software

PyroMark Q24 Advanced Software, installed on a PC, enables comprehensive analysis of your results. The software contains 4 analysis modes — AQ, SNP, CpG, and SEQ. The AQ mode can be used for a variety of quantification studies of mutations such as SNPs and InDels. It is suitable for analyzing single and multivariable positions, as well as di-, tri- , and tetra- allelic mutations. The SNP mode provides genotype analysis of SNPs and InDels. The CpG mode enables methylation analysis of single or multiple CpG or CpN sites and provides a built-in control for the bisulfite treatment. The SEQ mode is used for base-calling of unknown sequences.

The PyroMark Q24 Advanced Software is user-friendly and intuitive and provides convenient and improved tools for run analysis. If a problem occurs during the run, or if the system detects an inconsistency with an assay, the software provides specific warning information for each individual well (see Warning information and recommendations). A “Warning Info” button gives access to additional information about the warning along with recommendations for troubleshooting and preventing its occurrence in subsequent assays.

PyroMark Q24 Advanced Software version 3.0.1 is compatible with Windows 7 and Windows 10 (64 bit) operating systems. This software may only be downloaded by registered users with a valid PyroMark Q24 Advanced Software license and registered PyroMark Q24 Advanced instrument. If you do not have a valid software license, contact your QIAGEN sales representative.